Supply of proton enhances CO electrosynthesis for acetate and volatile fatty acid productions.

The microbial electrosynthesis is a platform to supply protons and electrons to improve the conversion efficiency and production rate for the valorization of C1 gas. This study examined proton migration and electron transfer of the electrode and microbe by using various external parameters in the electrosynthesis of CO. The CO electrosynthesis achieved almost double of coulombic efficiency than the conventional CO2 electrosynthesis. The maximum volumetric acetate production rate was 0.71 g/L/day in the BES, which was 2-6 times higher than reported elsewhere. These results show that the efficient proton migration and electron transfer can enhance the productivity and conversion efficiency of the biological CO conversion in a bioelectrochemical system.

Enabling anoxic acetate assimilation by electrode-driven respiration in the obligate aerobe, Pseudomonas putida.

This study examined the obligate aerobe, Pseudomonas putida, using acetate as the sole carbon and energy source, and respiration via an anode as the terminal electron acceptor under anoxic conditions. P. putida showed significantly different acetate assimilation in a closed-circuit microbial fuel cell (CC-MFC) compared to an open circuit MFC (OC-MFC). More than 72% (2.6 mmol) of acetate was consumed during 84 hrs in the CC-MFC in contrast to the no acetate consumption observed in the OC-MFC. The CC-MFC produced 150 µA (87 C) from acetate metabolization. Electrode-based respiration reduced the NADH/NAD+ ratio anaerobically, which is similar to the aerobic condition. The CC-MFC showed significantly higher acetyl-CoA synthetase activity than the OC-MFC (0.028 vs. 0.001 µmol/min/mg), which was comparable to the aerobic condition (circa 60%). Overall, electrode-based respiration enables P. putida to metabolize acetate under anoxic conditions and provide a platform to regulate the bacterial redox balance without oxygen.

Development of Klebsiella pneumoniae J2B as microbial cell factory for the production of 1,3-propanediol from glucose.

1,3-Propanediol (1,3-PDO) is an important platform chemical which has a wide application in food, cosmetics, pharmaceutical and textile industries. Its biological production using recombinant Escherichia coli with glucose as carbon source has been commercialized by DuPont, but E. coli cannot synthesize coenzyme B12 which is an essential and expensive cofactor of glycerol dehydratase, a core enzyme in 1,3-PDO biosynthesis. This study aims to develop a more economical microbial cell factory using Klebsiella pneumoniae J2B which can naturally synthesize coenzyme B12. To this end, the heterologous pathway for the production of glycerol from dihydroxyacetone-3-phosphate (DHAP), a glycolytic intermediate, was introduced to J2B and, afterwards, the strain was extensively modified for carbon and energy metabolisms including: (i) removal of carbon catabolite repression, (ii) blockage of glycerol export across the cell membrane, (iii) improvement of NADH regeneration/availability, (iv) modification of TCA cycle and electron transport chain, (v) overexpression of 1,3-PDO module enzyme, and (vi) overexpression of glucose transporter. A total of 33 genes were modified and/or overexpressed, and one resulting strain could produce 814 mM (62 g/L) of 1,3-PDO with the yield of 1.27 mol/mol glucose in fed-batch bioreactor culture with a limited supplementation of coenzyme B12 at 4 µM, which is ~10 fold less than that employed by DuPont. This study highlights the importance of balanced use of glucose in the production of carbon backbone of the target chemical (1,3-PDO) and regeneration of reducing power (NADH). This study also suggests that K. pneumoniae J2B is a promising host for the production of 1,3-PDO from glucose.

Development of 3-hydroxypropionic-acid-tolerant strain of Escherichia coli W and role of minor global regulator yieP.

3-Hydroxypropionic acid (3-HP) is an important platform chemical, but its toxic effect at high concentrations (> 200 mM) is a serious challenge for commercial production. In this study, a highly 3-HP-tolerant strain of Escherichia coli W (tolerance concentration: 400 mM in M9 minimal medium and 800 mM when yeast extract was added) was developed by adaptive laboratory evolution (ALE) with glycerol as the carbon source. Genome analysis of the adapted strain (designated as E. coli WA) indicated the presence of mutations in 13 genes, including glpK (glycerol kinase) and yieP (a less-studied global regulator). The mutant GlpK (K67T) exhibited a higher activity than the wild-type enzyme, but it was not beneficial for 3-HP production due to its causing carbon overflow metabolism. Interestingly, among the other 12 genes, the mutation in yieP alone was almost fully responsible for the improved tolerance to 3-HP. When the mutant yieP was substituted with the wild-type counterpart, the adapted E. coli WA strain completely lost its tolerance to 3-HP, showing a tolerance similar to the wild-type W strain. Deletion of yieP conferred 3-HP tolerance to several other E. coli strains including K-12 W3110, K-12 MG1655, and B except BL21 (DE3). The E. coli WA with wild-type glpK showed, as compared with its parental strain, better 3-HP production, indicating that improved tolerance is beneficial for 3-HP production.

Metabolic engineering of Klebsiella pneumoniae J2B for co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol: Reduction of acetate and other by-products.

Production of 3-hydroxypropionic acid (3-HP) or 1,3-propanediol (1,3-PDO) production from glycerol is challenging due to the problems associated with cofactor regeneration, coenzyme B12 synthesis, and the instability of pathway enzymes. To address these complications, simultaneous production of 3-HP and 1,3-PDO, instead of individual production of each compound, was attempted. With over-expression of an aldehyde dehydrogenase, recombinant Klebsiella pneumoniae could co-produce 3-HP and 1,3-PDO successfully. However, the production level was unsatisfactory due to excessive accumulation of many by-products, especially acetate. To reduce acetate production, we attempted; (i) reduction of glycerol assimilation through the glycolytic pathway, (ii) increase of glycerol flow towards co-production, and (iii) variation of aeration rate. These efforts were partially beneficial in reducing acetate and improving co-production: 21g/L of 1,3-PDO and 43g/L of 3-HP were obtained. Excessive acetate (>150mM) was still produced at the end of bioreactor runs, and limited co-production efficiency.

Production of 3-hydroxypropionic acid by balancing the pathway enzymes using synthetic cassette architecture.

Biological 3-hydroxypropionic acid (3-HP) production from glycerol is a two-step reaction catalyzed by glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH). Recombinant strains developed for 3-HP production often suffer from the accumulation of a toxic intermediate, 3-hydroxypropionaldehyde (3-HPA). In order to avoid 3-HPA accumulation, balancing of the two enzymatic activities, in the present study, was attempted by employment of synthetic-regulatory cassettes comprising varying-strength promoters and bicistronic ribosome-binding sites (RBSs). When tested in recombinant Escherichia coli, the cassettes could precisely and differentially control the gene expression in transcription, protein expression and enzymatic activity. Five recombinant strains showing different expressions for GDHt were developed and studied for 3-HPA accumulation and 3-HP production. It was found that 3-HPA accumulation could be completely abolished when expressing ALDH at a level approximately 8-fold higher than that of GDHt. One of the strains, SP4, produced 625mM (56.4g/L) of 3-HP in a fed-batch bioreactor, though late-period production was limited by acetate accumulation. Overall, this study demonstrated the importance of pathway balancing in 3-HP production as well as the utility of the synthetic cassette architecture for precise control of bacterial gene expression.

Metabolic engineering of Klebsiella pneumoniae J2B for the production of 1,3-propanediol from glucose.

The production of 1,3-propanediol (1,3-PDO) from glucose was investigated using Klebsiella pneumoniae J2B, which converts glycerol to 1,3-PDO and synthesize an essential coenzyme B12. In order to connect the glycolytic pathway with the pathway of 1,3-PDO synthesis from glycerol, i.e., to directly produce diol from glucose, glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatase from Saccharomyces cerevisiae were overexpressed. Additionally, the effect of expression levels and the use of isoforms of these two enzymes on glycerol and 1,3-PDO production were studied. Furthermore, to prevent loss of produced glycerol, the glycerol oxidation pathways were disrupted. Finally, the conversion rate of glycerol to 1,3-PDO was increased via homologous overexpression of glycerol dehydratase and 1,3-PDO oxidoreductase. The resultant strain successfully produced 1,3-PDO from glucose at a yield of 0.27mol/mol along with glycerol at 0.52mol/mol. Improvement of the engineered K. pneumoniae J2B to further increase conversion of glycerol to 1,3-PDO is discussed.

Citrobacter amalonaticus Y19 for constitutive expression of carbon monoxide-dependent hydrogen-production machinery.

BACKGROUND: Citrobacter amalonaticus Y19 is a good biocatalyst for production of hydrogen (H2) from oxidation of carbon monoxide (CO) via the so-called water-gas-shift reaction (WGSR). It has a high H2-production activity (23.83 mmol H2 g-1 cell h-1) from CO, and can grow well to a high density on various sugars. However, its H2-production activity is expressed only when CO is present as an inducer and in the absence of glucose. RESULTS: In order to avoid dependency on CO and glucose, in the present study, the native CO-inducible promoters of WGSR operons (CO dehydrogenase, CODH, and CODH-dependent hydrogenase, CO-hyd) in Y19 were carefully analyzed and replaced with strong and constitutive promoters screened from Y19. One engineered strain (Y19-PR1), selected from three positive ones after screening ~10,000 colonies, showed a similar CO-dependent H2-production activity to that of wild-type Y19, without being affected by glucose and/or CO. Compared with wild-type Y19, transcription of the CODH operon in Y19-PR1 increased 1.5-fold, although that of the CO-hyd operon remained at a similar level. To enhance the activity of CO-Hyd in Y19-PR1, further modifications, including an increase in gene copy number and engineering of the 5' untranslated region, were attempted, but without success. CONCLUSIONS: Convenient recombinant Y19-PR1 that expresses CO-dependent H2-production activity without being limited by CO and glucose was obtained.

Co-production of hydrogen and ethanol from glucose in Escherichia coli by activation of pentose-phosphate pathway through deletion of phosphoglucose isomerase (pgi) and overexpression of glucose-6-phosphate dehydrogenase (zwf) and 6-phosphogluconate dehydrogenase (gnd).

BACKGROUND: Biologically, hydrogen (H2) can be produced through dark fermentation and photofermentation. Dark fermentation is fast in rate and simple in reactor design, but H2 production yield is unsatisfactorily low as <4 mol H2/mol glucose. To address this challenge, simultaneous production of H2 and ethanol has been suggested. Co-production of ethanol and H2 requires enhanced formation of NAD(P)H during catabolism of glucose, which can be accomplished by diversion of glycolytic flux from the Embden-Meyerhof-Parnas (EMP) pathway to the pentose-phosphate (PP) pathway in Escherichia coli. However, the disruption of pgi (phosphoglucose isomerase) for complete diversion of carbon flux to the PP pathway made E. coli unable to grow on glucose under anaerobic condition. RESULTS: Here, we demonstrate that, when glucose-6-phosphate dehydrogenase (Zwf) and 6-phosphogluconate dehydrogenase (Gnd), two major enzymes of the PP pathway, are homologously overexpressed, E. coli Δpgi can recover its anaerobic growth capability on glucose. Further, with additional deletions of ΔhycA, ΔhyaAB, ΔhybBC, ΔldhA, and ΔfrdAB, the recombinant Δpgi mutant could produce 1.69 mol H2 and 1.50 mol ethanol from 1 mol glucose. However, acetate was produced at 0.18 mol mol-1 glucose, indicating that some carbon is metabolized through the Entner-Doudoroff (ED) pathway. To further improve the flux via the PP pathway, heterologous zwf and gnd from Leuconostoc mesenteroides and Gluconobacter oxydans, respectively, which are less inhibited by NADPH, were overexpressed. The new recombinant produced more ethanol at 1.62 mol mol-1 glucose along with 1.74 mol H2 mol-1 glucose, which are close to the theoretically maximal yields, 1.67 mol mol-1 each for ethanol and H2. However, the attempt to delete the ED pathway in the Δpgi mutant to operate the PP pathway as the sole glycolytic route, was unsuccessful. CONCLUSIONS: By deletion of pgi and overexpression of heterologous zwf and gnd in E. coli ΔhycA ΔhyaAB ΔhybBC ΔldhA ΔfrdAB, two important biofuels, ethanol and H2, could be successfully co-produced at high yields close to their theoretical maximums. The strains developed in this study should be applicable for the production of other biofuels and biochemicals, which requires supply of excessive reducing power under anaerobic conditions.

Measurement of crude-cell-extract glycerol dehydratase activity in recombinant Escherichia coli using coupled-enzyme reactions.

Glycerol dehydratase (GDHt), which converts glycerol to 3-hydroxypropionaldehyde, is essential to the production of 1,3-propanediol (1,3-PDO) or 3-hydroxypropionic acid (3-HP). A reliable GDHt activity assay in crude-cell extract was developed. In the assay, GDHt converted 1,2-propanediol (1,2-PDO) to propionaldehyde, which was further converted to 1-propionic acid by aldehyde dehydrogenase (KGSADH) or to 1-propanol by yeast-alcohol dehydrogenase (yADH), while the NADH concentration change was monitored spectrophotometrically. Cells should be disintegrated by Bead Beater/French Press, not by chemical methods (BugBuster®/B-PER™), because the reagents significantly inactivated GDHt and coupling enzymes. Furthermore, in the assay mixture, a much higher activity of KGSADH (>200-fold) or yADH (>400-fold) than that of GDHt should have been maintained. Under optimal conditions, both KGSADH and yADH showed practically the same activity. The coupled-enzyme assay method established here should prove to be applicable to recombinant strains developed for the production of 3-HP and/or 1,3-PDO from glycerol.

Co-production of hydrogen and ethanol by pfkA-deficient Escherichia coli with activated pentose-phosphate pathway: reduction of pyruvate accumulation.

BACKGROUND: Fermentative hydrogen (H2) production suffers from low carbon-to-H2 yield, to which problem, co-production of ethanol and H2 has been proposed as a solution. For improved co-production of H2 and ethanol, we developed Escherichia coli BW25113 ΔhycA ΔhyaAB ΔhybBC ΔldhA ΔfrdAB Δpta-ackA ΔpfkA (SH8*) and overexpressed Zwf and Gnd, the key enzymes in the pentose-phosphate (PP) pathway (SH8*_ZG). However, the amount of accumulated pyruvate, which was significant (typically 0.20 mol mol(-1) glucose), reduced the co-production yield. RESULTS: In this study, as a means of reducing pyruvate accumulation and improving co-production of H2 and ethanol, we developed and studied E. coli SH9*_ZG with functional acetate production pathway for conversion of acetyl-CoA to acetate (pta-ackA (+)). Our results indicated that the presence of the acetate pathway completely eliminated pyruvate accumulation and substantially improved the co-production of H2 and ethanol, enabling yields of 1.88 and 1.40 mol, respectively, from 1 mol glucose. These yields, significantly, are close to the theoretical maximums of 1.67 mol H2 and 1.67 mol ethanol. To better understand the glycolytic flux distribution, glycolytic flux prediction and RT-PCR analyses were performed. CONCLUSION: The presence of the acetate pathway along with activation of the PP pathway eliminated pyruvate accumulation, thereby significantly improving co-production of H2 and ethanol. Our strategy is applicable to anaerobic production of biofuels and biochemicals, both of which processes demand high NAD(P)H.

Co-production of hydrogen and ethanol from glucose by modification of glycolytic pathways in Escherichia coli - from Embden-Meyerhof-Parnas pathway to pentose phosphate pathway.

Hydrogen (H2) production from glucose by dark fermentation suffers from the low yield. As a solution to this problem, co-production of H2 and ethanol, both of which are good biofuels, has been suggested. To this end, using Escherichia coli, activation of pentose phosphate (PP) pathway, which can generate more NADPH than the Embden-Meyhof-Parnas (EMP) pathway, was attempted. Overexpression of two key enzymes in the branch nodes of the glycolytic pathway, Zwf and Gnd, significantly improved the co-production of H2 and ethanol with concomitant reduction of pyruvate secretion. Gene expression analysis and metabolic flux analysis (MFA) showed that, upon overexpression of Zwf and Gnd, glucose assimilation through the PP pathway, compared with that of the EMP or Entner-Doudoroff (ED) pathway, was greatly enhanced. The maximum co-production yields were 1.32 mol H2 mol(-1) glucose and 1.38 mol ethanol mol(-1) glucose, respectively. It is noteworthy that the glycolysis and the amount of NAD(P)H formed under anaerobic conditions could be altered by modifying (the activity of) several key enzymes. Our strategy could be applied for the development of industrial strains for biological production of reduced chemicals and biofuels which suffers from lack of reduced co-factors.

Inducible gene expression system by 3-hydroxypropionic acid.

BACKGROUND: 3-Hydroxypropionic acid (3-HP) is an important platform chemical that boasts a variety of industrial applications. Gene expression systems inducible by 3-HP, if available, are of great utility for optimization of the pathways of 3-HP production and excretion. RESULTS: Here we report the presence of unique inducible gene expression systems in Pseudomonas denitrificans and other microorganisms. In P. denitrificans, transcription of three genes (hpdH, mmsA and hbdH-4) involved in 3-HP degradation was upregulated by 3-HP by the action of a transcriptional regulator protein, LysR, and a cis-acting regulatory site for LysR binding. Similar inducible systems having an LysR transcriptional regulator were identified in other microorganisms that also could degrade 3-HP. A docking study showed that the 3-HP binding pocket is located between the N-terminal helix-turn-helix motif and the C-terminal cofactor-binding domain. CONCLUSIONS: This LysR-regulated 3-HP-inducible system should prove useful for control of the level of gene expression in response to 3-HP.

Complete genome sequence of novel carbon monoxide oxidizing bacteria Citrobacter amalonaticus Y19, assembled de novo.

We report here the complete genome sequence of Citrobacter amalonaticus Y19 isolated from an anaerobic digester. PacBio single-molecule real-time (SMRT) sequencing was employed, resulting in a single scaffold of 5.58Mb. The sequence of a mega plasmid of 291Kb size is also presented.

Carbon and energy balances of glucose fermentation with hydrogenproducing bacterium Citrobacter amalonaticus Y19.

For the newly isolated H2-producing chemoheterotrophic bacterium Citrobacter amalonaticus Y19, anaerobic glucose metabolism was studied in batch cultivation at varying initial glucose concentrations (3.5- 9.5 g/l). The carbon-mass and energy balances were determined and utilized to analyze the carbon metabolic-pathways network. The analyses revealed (a) variable production of major metabolites (H2, ethanol, acetate, lactate, CO2, and cell mass) depending on initial glucose levels; (b) influence of NADH regeneration on the production of acetate, lactate, and ethanol; and (c) influence of the molar production of ATP on the production of biomass. The results reported in this paper suggest how the carbon metabolic pathway(s) should be designed for optimal H2 production, especially at high glucose concentrations, such as by blocking the carbon flux via lactate dehydrogenase from the pyruvate node.